modification on lentiCRISPR V2

[huhaha]

Dear Zhang's labmembers,

I modified  lentiCRISPR V2 by replacing puro with EGFP.

when I did transfectio to produce virus, it looks working well, ~90% is GFP+.

But the virus cannot infect any cells, indicating I did not get any virus.

What is the possibility it could be?

thank you,

[Michael Peoples]

How did you clone in the eGFP?  

Are your transfected cells expressing the Cas9 protein as the same size as cells transfected with the original LentiCRISPR V2?

If you used the BamH1 site, did you include the P2A sequence (which starts at the BamH1 site) with the eGFP sequence?  

[mario]

Did you put a polyA after the GFP?
Just checking

[huhaha]

在 2015年5月9日星期六 UTC+2下午8:23:43，Michael Peoples写道：

How did you clone in the eGFP? 

 

1.I cut the lenticrispr v2 vector by using BamHI/MluI and purify the band ~14kb.

2. design PCR primer to amplify EGFP with P2A in front of it.

EGFPF: AGACGATGACGATAAGGGATCCGGCGCAACAAACTTCTCTCTGCTGAAACAAGCCGGAGATGTCGAAGAGAATCCTGGACCGATGGTGAGCAAGGGCGAGGAGC

EGFPR: GTAATCCAGAGGTTGATTGTCGACTTAACGCGTTTACTTGTACAGCTCGTCCATGCCGAG

3. purify PCR product and Gilbson ligation into backbone

How do you think this procedure? 

[huhaha]

Is it necessary to add polyA after GFP?

I check the sequence of lentiCRISPR v2, there is not polyA after puro.

在 2015年5月10日星期日 UTC+2下午10:43:31，mario写道：

[Michael Peoples]

I am sure that you did, but did you sequence downstream of your insertion?

Unfortunately, there are 2 MluI sites in the plasmid. You could have cut out the WPRE through the UTR.  Your GFP would be expressed, but it definitely would not integrate into the genome.  

 If you gel purified the digested plasmid before hand, did you notice a band around 9.7 kb, 4.9 kb and 231 bp?  

Looking at the plasmid about the only way that I can see to swap in your GFP using simple restriction enzyme digest and ligation is to use the BamH1 and BsiWI site.  Make sure to include a stop codon in the GFP.  You will have a spacer of ~500 junk nt between which is not ideal.

If you want to stick with the Mlu1 site, I would recommend trying a low conc. of the enzyme and digest for 3, 5, 10, 15 min.  You might get lucky and get some plasmids that don't cut at both sites.  But the fragment difference will be difficult to determine.  

Or if you are dead set on using this plasmid, you could always do a site directed mutagenesis by PCR targeting the other MLU1 site to kill it.  

In the end, it is always nice to have a fluorescent marker, but if you have done your puro kill curves on the target cells and use a conc. of puro that will kill the cells in ~3-4 days the antibiotic marker works well.

If you are having problems with transduction, you could concentrate your virus with a commercially available kit and that would help too.

Good Luck!

  

On Friday, May 8, 2015 at 5:56:01 PM UTC-5, huhaha wrote:

[mario]

Sorry, I was not clear in my previous post.
You should NOT add a poly A at the end of your gene, therefore you are fine in this regard
Ciao
Mario

[huhaha]

Thank you so much for the analysis. I am generating a mutation to kill one MluI.

By the way, could you send the catalog number of commercial available kit to concentrate virus?

best,
 

在 2015年5月13日星期三 UTC+2下午3:36:46，Michael Peoples写道：

[Michael Peoples]

We use 2 methods, either simple ultracentrifugation or the Lenti-X concentrator from clontech: 

http://www.clontech.com/US/Products/Viral_Transduction/Lentiviral_Transduction_Tools/Lentivirus_Concentration

 
Best of luck!

On Friday, May 8, 2015 at 5:56:01 PM UTC-5, huhaha wrote:

[huhaha]

thank you so much.

any difference in terms of virus recovery between these two method?

在 2015年5月15日星期五 UTC+2下午5:29:30，Michael Peoples写道：

[SI]

Hi,

does the lentiCRISPRv2 EGFP work? I would like to do FACS sorting rather than doing a selection with puromycin.

Best 

[Gasol]

Hi All,

I also cloned an NLS-GFP into the LentiCRISRP V2 vector. I just want to share my strategy in case anyone else is interested. I used BamH1 and RsrII (which cuts in the middle of the Puro resistance). I cloned in an NLS-GFP fragment that I amplified with the following ends: one with the BamH1+p2A and the other side with a stop codon+RsrII. Therefore, the new vector has again the Cas9 followed by a p2A and NLS-GFP with a stop codon. 

I haven't infected yet as I was cloning the sgRNA. I will do it shortly and inform how efficient the infection is.

Best,

Ana 

 

[cca]

Hi Ana,

did you manage to get the GFP into the LentiCRISRP V2 and can you infect your cells properly with it? When reading all the posts it looks as if it was not a trivial sub cloning. It would be great if you could share your experience (and maybe your construct?) with us!

Best

[KC]

Hi Ana,

Any updates on this plasmid?
Cheers
KC

[Mohammed Taha]

Hi
Did you get a good results about replacement of puromycin with GFP? Please, tell my because I use this vector to transduce NK cell lines (KHYG1 and NK92) and all cell die after selection with puromycin.

[Abigail Read]

Hi Ana,  

Were you able to get this cloning to work? I have been working on it for a couple of months without success. 

Thanks!

On Thursday, June 11, 2015 at 11:25:39 AM UTC-4, Gasol wrote:

[Naveen Kumar]

Dear Zhang lab members

We are developing Med23 knockout cells. We successfully cloned Med23 sequences (gRNA coding cDNA, 4 different targets) into pLentiCRISPR v2 to generate pLentiCRISPR v2.Med23.

Thereafter, we produced Lentiviral vector by transfecting  pLentiCRISPR v2.Med23, pCMV delta R8.2 and pVSV constructs in HEK 293T cells. These Lentiviral vectors were transduced in HeLa cells in presence of polybrene (2 ug/ml) for 48 h and then selected by puromycin (15 ug/ml) for next 48 h. However, all the cells died following puromycin treatment.

In a control Lentiviral vector reaction where pLentiCRISPR v2.Med23 was replaced with pHR-CMV-GFP, the pesudotyped viruses produced GFP in >80% transduced cells suggesting lentiviral production was OK.

Can you please help me obtain right puromycin-resistant clones?

Thanks

Sincerely yours

Naveen Kumar